Targeted therapy for intractable cancer on the basis of molecular profiles: An open-label, phase II basket trial (Long March Pathway)

Purpose We evaluated he effects of molecular guided-targeted therapy for intractable cancer. Also, the epidemiology of druggable gene alterations in Chinese population was investigated. Materials and methods The Long March Pathway (ClinicalTrials.gov identifier: NCT03239015) is a non-randomized, open-label, phase II trial consisting of several basket studies examining the molecular profiles of intractable cancers in the Chinese population. The trial aimed to 1) evaluate the efficacy of targeted therapy for intractable cancer and 2) identify the molecular epidemiology of the tier II gene alterations among Chinese pan-cancer patients. Results In the first stage, molecular profiles of 520 intractable pan-cancer patients were identified, and 115 patients were identified to have tier II gene alterations. Then, 27 of these 115 patients received targeted therapy based on molecular profiles. The overall response rate (ORR) was 29.6% (8/27), and the disease control rate (DCR) was 44.4% (12/27). The median duration of response (DOR) was 4.80 months (95% CI, 3.33−27.2), and median progression-free survival (PFS) was 4.67 months (95% CI, 2.33−9.50). In the second stage, molecular epidemiology of 17,841 Chinese pan-cancer patients demonstrated that the frequency of tier II gene alterations across cancer types is 17.7%. Bladder cancer had the most tier-II alterations (26.1%), followed by breast cancer (22.4%), and non-small cell lung cancer (NSCLC; 20.2%). Conclusion The Long March Pathway trial demonstrated a significant clinical benefit for intractable cancer from molecular-guided targeted therapy in the Chinese population. The frequency of tier II gene alterations across cancer types supports the feasibility of molecular-guided targeted therapy under basket trials.


Introduction
Oncogenic alterations could result in the promotion of carcinogenesis and cancer progression (1). Development of therapy targeting druggable mutations (potentially actionable with an already approved therapy) enables patients with these gene alterations to experience significantly improved clinical benefits compared to conventional therapy (e.g., targeting HER-2 overexpression in the breast or gastric cancer, BRAF V600E mutation in melanoma, EGFR or ALK in lung cancer) (2-4). The choice of therapy guided by an individual's tumor molecular profile has become the standard of care for these cancer types. It has been proposed that druggable gene alterations may occur across a wide variety of tumor types due to the advancements in sequencing technologies and a deeper understanding of gene alterations in different tumor types (5). The unanswered question is whether regimens that target specific gene alterations can induce responses regardless of the cancer histology, or location.
To this end, a novel clinical trial design referred to as a "basket trial", was developed. This study design focuses on specific gene alterations found in tumors, regardless of the location of cancer origins (6)(7)(8). Based on prior basket trials investigating the presence of specific gene alterations, a few of therapeutic strategies have been approved for pan-cancer patients independent of cancer types (i.e., larotrectinib for neurotrophic tyrosine kinase, receptor, type (NTRK) 1-3 fusion, pembrolizumab for mismatch repair high deficiency/ microsatellite instability, and pembrolizumab for high tumor mutation burden [TMB]; >10 mut/Mb]) (9)(10)(11).
Intractable cancers are challenging to treat since they do not respond to currently approved therapies. Also included under the broad umbrella of intractable cancers are advanced cancers that have been treated (unsuccessfully) with multiple lines of therapy, as well as those without an effective standard of care (including rare cancers) (12). There is early evidence to suggest that basket trials may provide more clinical benefits than best supportive care for intractable cancers. Hyman et al. (13,14) conducted a basket trial of 120 refractory cancer patients harboring BRAF mutations, all of whom received vemurafenib. In non-small cell lung cancer (NSCLC) patients, the objective response rate (ORR) was 42% after multiline therapy, which is a more favorable response when compared with an ORR of 7% reported for standard, second-line treatment with docetaxel. In a cholangiocarcinoma cohort, the disease control rate (DCR) was 62%, which was also better than second-line chemotherapy (49.5%) (15). In addition to single-gene alterations, basket trials targeting multiple gene alterations have also been conducted for intractable cancers (the MyPathway study) (16).
Previous basket trials have mainly focused on non-East Asian populations. It is known that gene alterations driving cancer progression differ significantly across ethnicities (17). Both the effects of targeted therapy on the basis of molecular profiles for intractable cancer and the molecular epidemiology of druggable gene alterations in the Chinese population remain unclear. The present study aimed to evaluate the efficacy of molecular-guided targeted therapy for intractable cancer among 520 patients from a single cancer center, then further to identify the molecular epidemiology of druggable gene alterations among another 17,841 pan-cancer patients from 24 cancer centers in China.
The druggable gene alterations were defined to be either tier I gene alterations or tier II gene alterations. Tier I gene alterations have the strongest clinical significance, and are supported by level A evidence (Food and Drug Administration [FDA]-approved therapy included in professional guidelines) and level B evidence (wellpowered studies with consensus from experts in the field). Tier II gene alterations are of potential clinical significance, and are supported by level C evidence (FDA-approved therapies for different tumor types or investigational therapy, multiple small published studies with some consensus) and level D evidence (preclinical trials or a few cases report without consensus) (18).

Materials and methods
Trial design and procedure The Long March Pathway (ClinicalTrial.gov identifier: NCT03239015) is a non-randomized, open-label, phase II trial that combines multiple basket studies of intractable pan-cancer patients from China. The trial was conducted in 2 main stages. The first part of the Long March Pathway trial included 2 steps. In the first step, 520 intractable cancer patients were recruited prospectively to identify druggable gene alterations. Intractable cancer patients, considered to be those patients who had no standard of care according to the National Comprehensive Cancer Network (NCCN) guidelines (i.e., those with advanced solid tumor after multiple-line therapy, those unresponsive to the standard of care, those ineligible for standard care because of poor performance status) older than 18 years with any type of metastatic solid tumor were eligible for this trial. Additional eligibility requirements included measurable, or evaluable, lesions (19).
Genetic profiling was determined using a next-generation sequencing (NGS) platform (Burning Rock Biotech, Guangzhou, China) in a clinical laboratory improvement amendment (CLIA)approved laboratory. The protocols were described previously (20,21). Briefly, genetic profiling was performed using tumor tissue from the most recent tumor biopsy or circulating tumor DNA (ctDNA). DNA was extracted and the NGS library was prepared. Target capture was performed using a panel consisting of 520 cancer-related genes (OncoScreen Plus 520) spanning 1.64 megabases of the human genome. Indexed samples were sequenced on Nextseq 500 (Illumina, Inc., CA, USA) with paired-end reads and an average sequencing depth of 1,000× for tissue samples and 10,000× for liquid biopsy samples. Sequencing data were mapped to the human genome (hg19) using Burrows-Wheeler aligner 0.7.10 (22). Tissue and plasma samples were compared against their own white blood cell control to identify somatic variants. Variants were filtered using the VarScan fpfilter pipeline. Base calling in plasma and tissue samples required at least 8 supporting reads for single nucleotide variations (SNVs) and 2 and 5 supporting reads for insertion-deletion variations (Indels), respectively. Single nucleotide polymorphisms (SNPs) were excluded if population frequency of was over 0.1% in the ExAC, 1000 Genomes, dbSNP or ESP6500SI-V2 databases. Copy number variations (CNVs) were analyzed based on the depth of coverage data of capture intervals.
Based on their options, patients with the above druggable gene alterations were able to receive a molecular-guided targeted therapy. Patients with ALK fusions, MET amplification, or ROS1 fusions were treated with crizotinib (250 mg orally twice daily). Patients with ERBB2 alterations were treated with pyrotinib (400 mg orally once daily). Patients with CDK4/6 amplifications or CDKN2A/2B deletion mutations were treated with palbociclib (125 mg orally once daily for 14 days every 3 weeks). Patients with BRCA1/2 mutations were treated with olaparib (400 mg orally twice daily). Patients with BRAF mutations were treated with vemurafenib (960 mg orally once daily). Patients with EGFR mutations were treated with a firstgeneration EGFR tyrosine kinase inhibitor (TKI; gefitinib 250 mg orally once daily). Patients with PIK3CA mutations were treated with everolimus (10 mg orally once daily). Patients with EGFR amplifications were treated with nimotuzumab (100 mg intravenous injection once per week). Patients with FGFR1/2/3 fusions or mutations were treated with erdafitinib (8 mg orally once daily). Patients received treatment for 2 cycles and were then evaluated for treatment response. Patients with objective response or stable disease (SD) continued the targeted therapy, with repeat evaluations every 2 cycles for the first 24 weeks, followed by evaluations every 12 weeks until tumor progression or unacceptable toxicity.
In the second stage of the trial, we conducted a multi-center study to determine the possibility of the above described molecular-guided targeted therapy in pan-cancer. The molecular epidemiology of the druggable gene alterations were evaluated in a larger cohort of 17,841 pan-cancer patients across 16 principal tumors types. In this stage, cancer types included NSCLC, SCLC, breast cancer, colorectal cancer, stomach cancer, liver cancer, pancreatic cancer, biliary system cancer, ovarian cancer, uterus cancer, prostate cancer, bladder cancer, kidney cancer, malignant melanoma, head and neck tumors, and sarcoma. All samples were obtained from fresh tissue or formalin-fixed paraffin-embedded tumor tissue, and they were profiled in a CLIAcertified NGS laboratory using the OncoScreen 295 or OncoScreen Plus 520 cancer-related gene panels (Burning Rock Biotech, Guangzhou, China). Molecular epidemiology of the tier II gene alterations in the pan-cancer patients were also determined based on genetic profiling. The trial was approved by the Ethics Committee of Changzheng Hospital (2017SL016). All participants provided written informed consent to take part in the study.

Study endpoints
The primary study endpoints for the Long March Pathway trial were ORR. Treatment response was evaluated by the investigator according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 (19). Secondary study endpoints included disease-control rate (DCR), duration of response (DOR), and progression-free survival (PFS). DOR was defined as the time interval from the first documentation of objective response to tumor progression/death, or the most recent tumor assessment. PFS was defined as the time from targeted therapy initiation to death from any cause or progression according to RECIST.
Among the cancer patients who underwent multiple-line therapy, PFS2/PFS1 ratios were also analyzed. PFS2 was defined as the PFS after genomics-guided targeted therapy. PFS on prior therapy (PFS1) was defined as the time interval from the most recent prior treatment to progression (as defined by RECIST), or clinical progression. A PFS2/PFS1 ratio of greater than 1.3 was considered indicative of a treatment benefit, in keeping with the definition of clinical benefit in basket trials (23). Additional secondary study endpoints for the trial were the frequencies of druggable gene alterations outside of tumorspecific approved targeted therapy (tier II gene alterations) among the Chinese pan-cancer patients.

Statistical analysis
We used a Simon's minimax 2-stage design with a type I error of 0.10 and 90% power to test the null hypothesis of an ORR of 10% or lower versus the alternative hypothesis of one 30% or higher (24). The regimen would be considered worthy of further investigation if 2 or more of 18 patients achieved partial response (PR) or complete response (CR), after which another 9 patients would be included. Analyses of ORR and DCR were performed based on the efficacy analysis of the population, which included noting those patients with measurable disease who 1) were treated with targeted therapy on the basis of molecular profiles and assessed for clinical response, or 2) had discontinued treatment for any reason (e.g., cancer-related death) before the first response evaluation. DOR analysis was conducted among patients who showed objective responses. The data cutoff date for interim efficacy analysis was September 1, 2020. The Kaplan-Meier curve was drawn to estimate median DOR/PFS with a 95% confidence interval (CI). The frequency of druggable gene alterations across a variety of cancers was defined as the portion of patients with druggable gene alterations who could receive off-label use of a molecular-guided targeted therapy. All statistical analyses were conducted in MedCalc software (MedCalc Software, Mariakerke, Belgium). A P value <0.05 was considered statistically significant.

Efficacy of molecular-targeted therapy
Among the 27 patients receiving targeted therapy on the basis of molecular profiles, 13 patients discontinued treatment due to cancerrelated death or poor performance status before the first response evaluation; the patients were identified as non-responders. No patient was dead due to the toxicity of targeted therapy. The median duration of follow-up was 8.3 months (range 2.4-55.0 months). Among the additional 14 patients who received response evaluations, 8 patients showed a partial response, and 4 patients showed stable disease. Overall, ORR was 29.6% (8/27) and DCR was 44.4% (12/27) (Figure 2A). Figure 2B showed the follow-up of all 27 patients, several responders have long survival, while median DoR was 4.80 months (95% CI, 3.33−27.2) ( Figure 2C), and median PFS was 4.67 months (95% CI, 3.33−9.50) ( Figure 2D). PFS1 was available in 22 patients, and 5 patients (22.7%) showed a PFS2/PFS1 ratio of >1.3 without progressive disease or death under matched targeted therapy during follow-up. Figure 2E showed an 89-year-old patient with metastatic rectal cancer harboring ERBB2 amplification received pyrotinib (400 mg po qd) and achieved partial response for 5.9 months.

Frequency of off-label gene alterations in pan-cancer patients
A total of 17,841 pan-cancer patients were included to assess the frequency of off-lable gene alterations ( Table 2). The recurrent somatic alterations across 16 common tumor types are presented in Figure 3A. Among these potential druggable alterations, EGFR, KRAS, and PIK3CA gene alterations were the most common, while CDK pathway (CDK4/6, CDKN2A/2B), FGFR1/2/3, BRAF, RET was uncommon across pan-cancer. The frequency of the 16 druggable gene alterations across 16 common tumor types were shown in Figure 3B. EGFR mutation, ALK fusion, ROS1 fusion, RET fusion was the most common in NSCLC patients. BRAF alteration were most observed in melanoma and colorectal cancer. KRAS mutations were observed in colorectal or pancreatic cancer patients, and PIK3CA mutations mainly occurred in breast cancer and uterine cancer. (Supplementary Files 2, 3). Figure 4 displays the frequency of tier I and tier II alterations in 17,841 pan-cancer patients. The frequency of tier I gene alterations in pan-cancer patients was 37.6% ( Figure 4A). NSCLC patients harbored the most tier I gene alterations (55.1%), followed by breast cancer (49.9%), malignant melanoma (27.9%), and colorectal cancer (9.1%). After the tier I gene alterations were excluded, only tier II alterations with the possibility of off-label use of a targeted drug in pan-cancer patients remained. Figure 4B shows the tier II gene alterations among 17,841 pan-cancer patients. Overall, the frequency of tier II alterations in pan-cancer patients was 17.7%, suggesting that 17.7% of pancancer patients had the potential to receive a molecular-guided targeted therapy. Bladder cancer had the most tier II gene

Discussion
The Long March Pathway was a basket trial designed to evaluate the efficacy of targeted therapy on the basis of molecular profiles for intractable cancer harboring tier II gene alterations. A total of 27 of 115 patients with tier II alterations received targeted therapy with an ORR of 29.6%, DOR of 44.4%, and PFS of 4.67 months. The molecular profiles of 17,841 pan-cancer patients showed that the frequency of tier II alterations across cancer types was 17.7%, indicating that 17.7% of patients could benefit from off-label molecular-guided targeted therapy.
Patients with intractable cancer do not respond well to standard chemotherapeutic regimens after multiple-line therapy, or cannot tolerate standard of care due to poor health conditions (12). In the era of genomics driving decision making in oncology, basket trials may provide guidance for the use of highly effective, and low-toxicity targeted therapy for these patients. However, there are two factors that warrant consideration prior to the application of targeted therapy to intractable cancer. First, it must be determined whether intractable cancer patients harbor specific druggable molecular alterations, especially for tier II gene alterations that do not have a currently available FDA indication. Second, it must be determined whether a molecular-guided targeted therapy will still be efficacious in intractable cancers with specific gene alterations.
To address the first point, the investigators have previously performed several studies. The ProfiLER study showed that 52% patients had at least 1 actionable mutation across 16 refractory cancer types (25). Similarly, SHIVA study found that 40% of refractory cancer patients had at least 1 druggable molecular alteration (26). The MOSCATO-01 study demonstrated that 411 out of 843 (49%) patients with advanced hard-to-treat cancers, and 42 out of 69 (60.9%) pediatric patients had actionable molecular alterations (27,28). With respect to rare cancers, Kato et al. (29) reported that 37 patients (92.5%) had at least 1 potentially actionable target among 40 patients who underwent genomic and protein analyses (30). Meanwhile, the genetic profiling of 5,954 refractory malignancies from the National Cancer Institute Molecular Analysis for Therapy Choice (NCI-MATCH) trial showed that 37.6% cases had an actionable alteration. Although there are some discrepancies among the reported frequencies, all these data demonstrated the existence of druggable molecular alterations among intractable cancers. As previous studies reported, the genetic landscape, and response to targeted therapy, vary across ethnicities (31). The molecular profiles of Chinese cancer patients remain unclear. Consistent with previous studies, this study demonstrated that 22.1% (115 of 520) of Chinese intractable cancer have at least 1 tier II alteration. Collectively, these data indicate the feasibility of molecular-guided targeted therapy and To further explore the possibility of molecular-guided targeted therapy across cancer types, the mutational landscape of metastatic cancer was analyzed using an NGS panel (256 or 520 gene panel) in 17,841 Chinese pan-cancer patients to detect the frequencies of druggable targets. The frequency of tier II gene alterations across pan-cancer outside of current FDA indications was 17.7%, suggesting that clinicians or physicians can consider various molecular targeted therapies across different tumor types with shared genetic features for the treatment of patients, especially for intractable cancer. In 2017, the mutational landscape of metastatic advanced cancer in 10,366 patients from the Memorial Sloan Kettering Cancer Center (MSKCC) was published. The study demonstrated that 27% of patients had at least 1 actionable alteration apart from targets with levels 1 and 2A evidence according to whether mutations were FDArecognized biomarkers. On comparing the distribution of actionable alterations between the MSCKCC and Long March Pathway cohorts, it was evident that while gynecological cancer and biliary cancer in the Chinese population harbored fewer alterations, lung cancer patients had more alterations than the MSKCC cohort. Some types of cancer, including breast cancer and bladder cancer, showed higher frequency of druggable targets in both cohorts Similarly, some gene alterations, including KRAS and PIK3CA mutations, were shared among different ethnicities, while some alterations, including EGFR mutation (enriched in Chinese NSCLC patients) were variably represented. This variation in the distribution of gene alterations thus warrants further investigations in other ethnic populations.
Previous studies have shown that targeted therapy may elicit different responses among patients from different histology. For example, melanoma patients harboring BRAF V600E were found to respond well to vemurafenib, while those with colorectal cancer did not (32,33). Similarly, recent trials have shown that colorectal cancer and NSCLC seem to respond differently to AMG510 although these cancers share KRAS G12C (34). Thus, whether targeted therapy, regardless of histology, are still efficacious in intractable cancers according to basket trials is yet to be fully elucidated, and controversies regarding the role of targeted therapy decisions based on genomics in intractable cancers warrants further investigation. Some studies have provided validation for this strategy in clinical practice, and that molecular-targeted cancer therapy based on basket trials may yield survival benefits in patients with intractable cancers (13,14,23,25,27,35). In addition, molecular-targeted cancer therapy has also resulted in improvements in prognosis among patients with rare cancers with no recommended regimen, younger cancer patients, and patients with refractory cancers of unknown origin (30,36,37). However, findings from some studies evaluating the efficacy of genomics-guided targeted therapy in intractable cancer are conflicting. The NCI-MATCH (EAY131) trial was the largest multiple basket trial study conducted to date that aimed to determine whether specific targeted therapies may benefit a greater number patients and lead to more personalized therapies in advanced cancer patients without standard treatment options (38). The EAY131-H study demonstrated an ORR of 38% after treatment of 29 pretreated, pan-cancer patients with mixed histology with dabrafenib and trametinib (39). However, the EAY131-Q study evaluated the efficacy of Ado-trastuzumab emtansine (T-DM1) in heavily pre-treated pan-cancer patients harboring HER2 amplification. The study noted an ORR rate of just 5.6% (40). The EAY131-W study found that the use of AZD4547, an oral FGFR1-3 inhibitor in patients with refractory cancer harboring aberrations in the FGFR pathway did not meet the primary endpoint (response rate of 16%) (41). Furthermore, the SHIVA study also demonstrated that the use of molecular-targeted therapy outside the approved indications did not significantly improve PFS in heavily treated cancer patients (42). There are several factors that may explain why this controversy regarding whether the use of genomics-guided therapy can improve outcomes in intractable cancer, including differences in targeted agents, oncogenic driving capacity, tumor heterogeneity, tumor genetic background, etc. (43)(44)(45). Although a common set of driver mutations exist within each cancer type, the combination of drivers and their distribution within the founding clone and subclones vary across patients, which could affect the efficacy of targeted therapy. This suggests that knowledge of the tumor's clonal architecture is crucial for optimizing treatment.
Meanwhile, the genetic backgrounds across different ethnicities may affect tumor-related molecular alterations. Molecular alteration frequency and response to targeted therapy also vary among across different ethnicities. Thus, the Long March Pathway trial conducted in the Chinese population is important and supports further genomics-guided targeted therapy in intractable cancer.
Our results have validated the use of targeted therapy on the basis of molecular profiles in clinical practice and have shown that the discussed methods can help guide treatment choices for intractable cancer. However, several limitations require clarification for accurate interpretation of the results. First, the ratio of patients who received off-label targeted therapy was low, and 23.5% (27 of 115) of patients received targeted therapy based on an individual molecular profile. The low ratio may be attributed to the high cost of targeted therapy and off-label treatment, which medical insurance does not cover. Second, a few of patients (48.1%, 13/27) failed to undergo response evaluation. Most patients discontinued the targeted therapy due to poor performance or cancer-related death during treatment. These patients were defined as non-responders to off-label targeted therapy. This might have affected the accuracy of efficacy evaluation. Third, the small sample size limited the efficacy evaluation of different druggable targets. For example, only 8 targeted therapies were assessed under multiple basket trials. The efficacy of molecular-targeted therapy against another 5 druggable targets (BRAF mutations, FGFR alterations, RET fusions, and ROS1 fusions) could not be evaluated, while the association of targeted agents type and the concomitant alterations with a response to targeted therapy were also not further assessed (46). In addition, the treatment regimen was not the current optimal standard care (e.g., crizotinib for ALK fusion, single-agent vemurifinib for BRAF V600E). Thus, the further exploration of the application of basket trials in a larger patient cohort is recommended.
In conclusion, the Long March Pathway trial demonstrated a significant clinical benefit for intractable cancers using targeted therapy based on molecular profiles in the Chinese population. The frequency of tier II gene alterations across a variety of cancers supports the feasibility of molecular-guided targeted therapy under basket trials and may provide novel insights into the optimal clinical treatment of intractable cancers.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author. Tier I or tier II alterations in 17,841 pan-cancer patients. (A) Tier I variants are of strongest clinical significance, and include level A evidence (FDAapproved therapy included in professional guidelines) and level B evidence (well-powered studies with consensus from experts in the field). (B) Tier II alterations reflect the possibility of off-label use of a targeted drug in pan-cancer patients. A tier II variant was defined as a variant of potential clinical significance, and included level C evidence (FDA-approved therapies for different tumor types or investigational therapy, multiple small published studies with some consensus) and level D evidence (preclinical trials or a few cases reports without consensus). FDA, food and drug administration.

Ethics statement
The studies involving human participants were reviewed and approved by The Ethics Committee of Changzheng Hospital. The patients/participants provided their written informed consent to participate in this study.